Dot matrix membrane cell expansion and maintenance vessel

ABSTRACT

There is disclosed a perfusable cell culture device capable of expansion and maintenance of biological cells, including mammalian, microbial, plant and insect cells in culture. The device comprises an upper and lower polymeric film layer and a gas and liquid permeable flow divider membrane. The flow divider membrane is affixed between the upper and lower polymeric layers and to the lower polymeric layer in a manner providing channels for fluid distribution of culture medium which is perfused at a controlled rate, uniformly across the flow divider membrane and to a bed of cells dispersed on the flow divider membrane.

BACKGROUND OF THE INVENTION

This invention relates to perfusable cell culture device, and moreparticularly comprises a flow divider membrane attached to a lowerpolymeric layer in a manner which provides channels for uniform fluiddistribution over the entire surface of the flow divider membrane andinto a bed of cells dispersed thereon.

Early in the history of tissue culture a need to perfuse culture systemswas recognized. As early as 1912, attempts to furnish cultures of animalcells with a continuous supply of fresh nutrient medium were made.Feder, J. , Tolbert, W. R. Mass Culture of Mammalian Cells in PerfusionSystems, ABL, Jan./Feb. 1985, p. 24-36. Since these early attempts,development of perfusion systems has continued and advancedsignificantly. For a good analysis of the development of perfusedsystems used for examination of mammalian cells see: Nahapetian, A. T.(1986). "Growth and Maintenance of Anchorage Dependent Mammalian Cellsin Perfused Systems and Metabolism of Nutrients", Chapter 7, MammalianCell Technology, W. G. Thilly(ed), pp. 151-165, Buttersworth, Boston.

Conventional cell culture systems incorporate a membrane or membranesused to diffuse either nutrients, metabolic waste, gases or cellularproducts into or out of the cell's environment. For example, U.S. Pat.No. 4,308,351 Leighton et. al. describes an apparatus comprisingencasing a tissue sample attached to a membrane and a support for themembrane, in a specially designed closed container filled with solutionand then bathed in a nutrient solution. Due to a designed nutrientconcentration gradient occurring in the tissue, the tissue samplereceives oxygen and nutrients by diffusion through the membrane from thenutrient bath as the membrane is exposed to the bath through aperturesin the closed container. U.S. Pat. No. 3,911,140 Osborne et. al.describes a fermenter in which a dialysis membrane separates a culturemedium containing propagating microorganisms from a regeneratingsolution containing nutrients. The dialysis membrane is impermeable tothe microorganisms and allows diffusion of desired nutrients based on aconcentration gradient of nutrients and lactic acid on either side ofthe membrane. Contrary to the conventional art, the present inventionincorporates a flow divider membrane which due to the manner in which itis affixed to the lower polymeric layer, allows for uniform distributionof culture medium over the entire flat surface of the flow dividermembrane, rather than incorporating a filter to control diffusion ofnutrients, metabolic waste, gases or cellular product between varyingsides of a concentration gradient.

The present invention provides a device designed to perfuse cells duringculture which is capable of providing a favorable environment forexpansion and maintenance of both anchorage dependent (in combinationwith microcarriers) and anchorage independent cells, such device havingthe following characteristics: 1) it provides continuous supply ofnutrients and continuous removal of metabolic end products; 2) itprovides adequate O₂ and CO₂ exchange with the surrounding environment;3) it maintains a favorable pH gas exchange; 4) it is designed such thatthe nutrients flow uniformly through the cell bed, independent ofdiffusion gradients or thermal agitation; 5) it is transparent therebyallowing visual examination of the culture and additionally allowinglight to pass through the vessel thereby providing an essentialenvironment when the device is used for plant cells; 6) it equilibratestemperature easily with the surrounding environment; 7) it exerts noshear, since there is no need for constant mixing of the culture; 8) itprovides a sterile environment; 9) it is non-breakable, disposable orsterilizable by autoclave; 10) it is not bulky and can be stacked one ontop of another when not in use, therefore conserving space; and 11) itis more cost effective than other perfused vessels available on themarket.

SUMMARY OF THE INVENTION

The invention is a disposable, sterile, plastic container useful as aperfusable cell culture device for biological cells including mammalian,microbial, plant and insect cells in culture. The device is structuredsuch that there are two liquid impermeable polymeric sheets, eachcapable of transferring oxygen and carbon dioxide at rates sufficient tomaintain cells in culture, hereinafter referred to as the upper andlower polymeric layers, which are joined together at their peripheraledges in a sealing manner. Between the upper and lower polymeric layersand affixed to the lower polymeric layer is a gas and liquid permeableflow divider membrane. This flow divider membrane is affixed to thelower polymeric layer with an evenly spaced pattern of seals. The twoare affixed such that the pressure drop across the flow divider issignificantly greater than the pressure drop through the channelscreated between the seals. Thus, substantially even pressure is providedacross the flow divider membrane, allowing culture medium to flowuniformly over a large flat surface area. The culture medium isintroduced into these channels at a controlled rate and uniformly flowsthrough the channels and over the entire surface of the cell dividermembrane. The device provides for culture medium to be introducedthrough an inlet port in the lower polymeric layer. The culture mediumflows between the lower polymeric layer and the flow divider membranethrough the channels for fluid distribution and into a chamber createdbetween the upper polymeric layer and the flow divider membrane. Thecells to be cultured are dispersed across the surface of the flowdivider membrane and are perfused at a controlled rate with culturemedium. A port in the upper polymeric layer acts as a means to placecells into the device, a means to remove cultured cells, and a means forcollecting perfused nutrients from the device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plane view schematically showing a preferred embodiment of adot matrix pattern of seals joining the lower polymeric layer and theflow divider membrane.

FIG. 2 is an enlarged schematic cross-section of the cell culturedevice.

FIG. 3 is an enlarged cross sectional view of the flow divider membraneshown in FIG. 2 with channels for fluid distribution.

FIG. 4 is a plane view schematically showing an alternate embodiment ofan evenly spaced pattern of seals joining the lower polymeric layer andthe flow divider membrane.

DETAILED DESCRIPTION

The flow divider membrane 1 shown in FIGS. 1, 2 and 3 made of a gas andliquid permeable material is joined to a lower polymeric layer 3 shownin FIG. 2, preferably in a dot matrix pattern 2 shown in FIGS. 1, 2 and3. As viewed in the cross-sectional positions of FIGS. 2 and 3 the sealsof the dot matrix pattern 2, used to join the flow divider membrane 1 tothe lower polymeric layer 3, provide channels for fluid distribution 6.Access of culture medium 11, to the channels for fluid distribution 6,is provided through the inlet port 4.

The upper and lower polymeric layers, 8 and 3 respectively, are made ofmaterial which is liquid impermeable and permeable to oxygen and carbondioxide at rates sufficient to maintain cells in culture. The peripheraledges 5 of the upper and lower polymeric layers, 8 and 3 respectively,are joined in a sealing manner.

As viewed in FIG. 1, a dot matrix pattern 2, in which the flow dividermembrane 1 is attached to the lower polymeric layer 3, preferably has amean pore size between 0.45 μm and 1.0 μm in diameter.

The flow divider membrane 1, is preferably made of expandedpolytetrafluoroethylene (PTFE) material, commercially available asGore-tex®. Uniform distribution of fluid through the flow dividermembrane 1 occurs because the pressure drop over the surface of the flowdivider membrane 1 is significantly greater than the pressure throughthe channels 6, which are formed by the seals of the dot matrix pattern2. The significantly greater pressure drop across the surface of theflow divider membrane 1, compared to the pressure in the channels forfluid distribution 6 allows for even pressure across the entire surfaceof the flow divider membrane and therefore the culture medium isuniformly distributed across the flow divider membrane. As used herein,uniformly means that the center and the outer edge or any other locationon the flow divider membrane are to get the same flow rate per unitarea, with no gross variation. By affixing, by thermal bonding, the flowdivider membrane to the lower polymeric layer in a dot matrix or asimilar evenly spaced pattern, the desired flat geometry between theflow divider membrane and the lower polymeric layer is achieved, [placesthe spaces between the dot matrix seals at essentially tensile load,thereby maintaining a substantially flat surface. As used herein tensileload means the greatest perpendicular stress a substance can bearwithout tearing apart.]The closer the seals are to each other in thepattern [the closer to tensile load the surface becomes and therefore]the flatter the surface is maintained.

In use, the cells to be cultured are placed onto the surface of the flowdivider membrane 1, through a port 9 in the upper polymeric layer 8.Culture medium is then perfused through the port 4 in the lowerpolymeric layer, into the channels for distribution 6 which provide foruniform distribution of the culture medium over the entire surface ofthe flow divider membrane 1 and through the bed of cells 10. Spentculture medium and products of culture can then be removed through theport 9 in the upper polymeric layer 8. A sterile environment must bemaintained within the device therefore a sterile connection system suchas Sterile Connection Device™, commercially available from E. I. DuPontde Nemours Co.,is preferably used at the port in the upper polymericlayer, connecting tubing leading from the port to additional tubing,syringe or other object used for adding cells, withdrawing cells orspent culture medium. Such a connection device allows for manipulationof the cell culture device without losing the sterile environment withinthe device.

A preferred embodiment of this invention includes using expanded PTFE,commercially available as Gore-Tex®, with a pore size between 0.45micron and 1.0 micron and preferably 0.45 micron, for the flow dividermembrane. Untreated PTFE material is highly hydrophobic to the extentthat 0.45 micron pore size untreated PTFE material will pass nomeasurable volume of aqueous 25 solution with a pressure differential of5 PSI. Treating the PTFE with a water soluable polymer, such aspolyvinylpyrroidone (PVP), solubilized in methanol or other wettingagent, decreases the water contact angle and permits the largest poresto be accessed when if untreated they would be inaccessible to water.Since the PTFE is hydrophobic and the PVP is hydrophilic, it isreasonable to expect a certain amount of PVP or other water solublepolymer, deposited on the PTFE fibers would leave the material weaklyhydrophobic or more hydrophilic so as to lessen water entry pressure.Treating PTFE with a specific concentration of PVP/methanol allows anaqueous flow of approximately 200 micro liters per minute at 5 PSI on asurface area of approximately 15 square inches with a pore size of 0.45micron. A greater concentration of PVP/methanol provides for increasedflow at a constant 5 PSI or decreased pressure at 200 micro liters perminute. The effects of flow and pressure changes with expanded PTFE arethe result of surface tension changes and not changes in pore size.Increased pressure with the same concentration of PVP/methanol accessessmaller pores for increased flow. Furthermore, increased concentrationsof PVP/methanol provide a lower surface tension of smaller pores forincreased flow at a constant pressure. Thus a further preferredembodiment of this invention would include taking advantage of thePVP/Methanol affect by tailoring a particular flow divider pore size todivide evenly (via a desired pressure drop), with a desired flow rate.For example 0.45 micron PTFE material can be treated with varyingconcentrations of PVP/Methanol for 200 micro liter flow at 3 PSI or 500micro liter at 3 PSI. Although expanded PTFE is a preferred material forthe flow divider membrane, other microporus membrane materials such asNucleopore® may be used. Nucleopore® , like expanded PTFE, is madehydrophilic by treatment with a solution of PVP or other water solublepolymer and methanol or other wetting agent.

A preferred method of treating expanded PTFE fibers such as Gore-Tex®,comprises solubilizing PVP powder in methanol which serves as a wettingagent and vehicle for the PVP into the expanded PTFE fibers. ThePVP/methanol solution is then applied to the expanded PTFE fibers, thisapplication can be by immersion or spraying while allowing for airdrying of the methanol. Preferrably, the application is by controlledimmersion and forced air drying such as by mechanically driven rollers.After drying, an amount of PVP will deposit on the expanded PTFE fibersleaving the material weakly hydrophobic or more hydrophilic. Once thePTFE fibers are made more hydrophilic, water can penetrate the material,this condition will remain as long as water is present and the materialis not allowed to dry out. The designed flow characteristics of thePVP/methanol treated expanded PTFE fibers will remain after the watersoluble PVP has solubilized and flushed away.

Preferred embodiments of this invention include using a PVP/methanolsolution particularly since PVP is relatively innocuous to cells used inthe device. However, other water soluble polymers such as polyethyleneglycol, polyvinyl alcohol or hydroxyethylmethacrylate are useful torender microporous membrane material such as expanded PTFE material morehydrophilic. Methanol is a preferred wetting agent/vehicle for the watersoluble polymer. Other wetting agents/vehicles such as ethanol,isopropyl alcohol, acetone, water, glycols or chlorinated solvents areuseful.

A preferred embodiment of this invention includes thermally bonding theflow divider membrane to the lower polymeric layer. A further preferredembodiment of this invention includes thermally bonding the flow dividermembrane to the lower polymeric layer in a dot matrix pattern. Althoughthermal bonding is preferred, other methods of affixing the flow dividermembrane to the lower polymeric layer such as bonding with adhesive ormechanically staking the two, can be used.

The rate of liquid flow through the device is important to thisinvention. Uniform flow division is conditional on having appropriateliquid flow to provide the selected back pressure across the populationof cells of interest, while not over pressurizing the flow divider andpackaging seals. If the back pressure is not attained, the liquid willbias through the center region of the divider and not flow uniformlyacross the divider. An embodiment of this invention includes liquid flowof between 100 and 500 micro liters per minute when a 5 inch diametervessel is used. Liquid flow rate will vary based on the unit area of thevessel used.

Polymeric film material suitable for constructing the cell culturedevice should be liquid impermeable and permeable to oxygen and carbondioxide at rates sufficient to maintain cells in culture, such as thoseset forth in U.S. Pat. No. 4,588,401 issued on May 13, 1986 to Kilkson.Suitable polymeric film material can be selected from but not limited tothe group consisting of a copolymer of ethylene and an alpha olefin,such as Sclairfilm®, polytetrafluoroethylene, commercially available asTeflon®, fluorinated ethylenepropylene copolymer (FEP) also available asTeflon®, polyester elastomers, such as Hytrel® commercially availablefrom E. I. du Pont de Nemours.

Further embodiments of this invention include the addition of one ormore additional membranes, such as size exclusion membranes, to beaffixed to the upper polymeric layer of the device so that internalseparations can take place in order to isolate products which may beproduced during cultivation. Uniform flow of culture medium wouldcontinue to occur across the flow divider membrane independent ofdiffusion gradients although such diffusion gradients may be presentacross the surface of additional membranes optionally affixed to theupper polymeric layer. Any additional membranes affixed to the upperpolymeric layer would optionally have separate tubing leading from theadditional membrane to an additional outlet port in the upper polymericlayer. This additional tubing and additional outlet port would allow forcollection of the separated material.

Further embodiments of this invention include the presence of a pumpsuch as a Multistaltic Pump, commercially available from Haake Buchler,which ideally can pump a settable volume of flow per minute. Such a pumpis connected to the inlet port in the lower polymeric layer by tubing.Such tubing may also be connected to the port in the upper polymericlayer. A preferred embodiment of this invention includes a connectiondevice, such as the Sterile Connection Device™ commercially availablefrom E. I. Du Pont, used at the port in the upper polymeric layer whenadding cells, withdrawing culture medium, removing cells or performingother manipulations on the device which could cause loss of the sterileenvironment. The pump and tubing used in the present invention may be ofa known type. In addition, the manner in which this tubing is connectedto the port on either the upper or lower layer of the cell culturedevice may be of a manner known in the art.

The perfusable cell culture device of the present invention provides anovel environment for expansion and maintenance of both anchoragedependent (in combination with microcarriers) and anchorage independentbiological cells, including mammalian, microbial, plant and insectcells. The present invention is designed to achieve the highest possibledensity of cells. A high number of cells can be cultured using thisdevice since the device perfuses oxygen as well as nutrients in theculture medium through the bed of cells. The present invention isparticularly useful for culturing mammalian cells such as hybridomacells (HB47), U937 cells and more particularly tumor infiltratinglymphocytes (TIL) cells and lymphokine activated killer (LAK) cells.Culture medium used in the present invention may be of a known type.

The following examples further illustrate the usefulness of thisinvention for the cultivation of cells.

EXAMPLE 1

A cell culture vessel was structured with a Gore-Tex® expanded PTFE flowdivider membrane heat sealed by use of a dot matrix pattern to acopolymeric film of polyethylene and linear low density polyethylene,commercially available from DuPont Canada as Sclairfilm® said expandedPTFE material being first treated with PVP solubized in methanol.Application of this solution was in the form of immersion and sprayingwhile allowing for air drying of the methanol. A more preferred andconsistent method of application includes forced air drying andcontrolled immersion such as by mechanically driven rollers.

A concentration of 1 gram PVP to 30 ml methanol yields effectivepressure and flow.

The dry expanded PTFE Gore-Tex®, 0.45μ mean pore size, flow dividermembrane was bonded to the copolymeric film in a dot matrix pattern bymeans of a textured heated platen at approximately 300 degrees F., forapproximately 5 seconds at approximately 30 PSI. The textured surface,having 0.06 diameter raised dots at 0.125 centers, provides physicalconnection of the two materials and also channels for fluid distributionbetween the materials.

Completion of the device less further embodiments and ancillaryconnector fittings was accomplished by connecting an upper polymericlayer to the previously affixed flow divider membrane and lowerpolymeric layer, at their peripheral edges. Bonding temperature,pressure, and dwell time is dependent on the materials used. Forexample, when fluorinated ethylenepropylene copolymer (FEP) commerciallyavailable as Teflon® is used as the upper and lower polymeric layer, thePTFE and FEP bonding temperature is approximately 700 degrees F., forapproximately 10 seconds at approximately 40 PSI.

The entire device is schematically represented in FIG. 2.

EXAMPLE 2

The 0.45μ vessel of Example 1 was used for the expansion and maintenanceof a hybridoma which produces monoclonal antibody to interleukin -2(IL-2). The dot matrix perfused system was maintained by having freshIscove's modified DMEM medium perfused at a rate of 250 ml/day. Theperfused system was maintained in an incubator containing 5% CO₂ in airat 37° C. Cultures were compared to available fed-batch controlSteriCell™ cell culture bags (DuPont) having a growth surface area of150 cm², which were maintained in the same environment. The followingdata were taken:

    ______________________________________                                                         Dot-matrix                                                                    Perfused                                                                              Fed Batch                                            ______________________________________                                        Total viable cells 1.54 × 10.sup.7                                                                   1.54 × 10.sup.7                            (time = 0)                                                                    Total viable cells 1.05 × 10.sup.9                                                                   2.78 × 10.sup.8                            (time = 7 days)                                                               Cell viability     85%       85%                                              (time = 0)                                                                    Cell viability     90%       77%                                              (time = 7 days)                                                               Viable cell number/cm.sup.2                                                                      1.25 × 10.sup.5                                                                   1.23 × 10.sup.5                            (time = 0)                                                                    Viable cell number/cm.sup.2                                                                      8.54 × 10.sup.6                                                                   1.85 × 10.sup.6                            (time = 7 days)                                                               Viable cell number/ml                                                                             2.9 × 10.sup.5                                                                    2.9 × 10.sup.5                            (time = 0)                                                                    Viable cell number/ml                                                                             1.5 × 10.sup.7                                                                   1.39 × 10.sup.6                            (time = 7 days)                                                               Number of monocell layers                                                                        0.1       0.1                                              (time = 0)                                                                    Number of monocell layers                                                                        6.83      1.48                                             (time = 7 days)                                                               ______________________________________                                    

The data show enhancements of perfused versus fed-batch cultures intotal cells of 378%, cell viability, 117%; cell number/cm 461%; viablecell number/ml, 1079%; and number of monocell layers 461%.

EXAMPLE 3

U937 cells were used for a comparative study of the cell expansion in a0.45μ dot-matrix perfused vessel of Example 1 and a fed-batch SteriCell™system (DuPont). The following data were collected:

    ______________________________________                                        U937 CELLS                                                                                       Perfused                                                                      Dot-Matrix                                                                            Fed-Batch                                          ______________________________________                                        Growth surface area, CM2                                                                           123       150                                            Total viable cell number on day 0                                                                  8.05 × 10.sup.7                                                                   2.28 × 10.sup.8                          Total viable cell number on day 24                                                                 8.91 × 10.sup.8                                                                   5.12 × 10.sup.8                          Cell viability on day 0                                                                            69%       77%                                            Cell viability on day 24                                                                           84%       59%                                            Viable cell number per CM2 on day 0                                                                7.12 × 10.sup.5                                                                   1.52 × 10.sup.6                          Viable cell number per CM2 on day 24                                                               7.88 × 10.sup.6                                                                   3.41 × 10.sup.6                          Viable cell number per ml on day 0                                                                 9.70 × 10.sup.5                                                                   1.14 × 10.sup.6                          Viable cell number per ml on day 24                                                                1.31 × 10.sup.7                                                                   2.56 × 10.sup.6                          Number of mono-cell-layers on day 0                                                                0.57      1.22                                           Number of mono-cell-layers on day 24                                                               6.31      2.73                                           ______________________________________                                    

The data show enhancements of perfused versus fed-batch cultures intotal viable cell number 180%, viable cell number/cm² 400% monocelllayers 250%, viability (%) 142%, viable cell number/ml 5000%.

EXAMPLE 4

TIL 714 cells were used for a comparative study of the cell expansionand maintenance in a 0.45μ dot-matrix perfused vessel of Example 1 and afed-batch SteriCell™ system (DuPont). The following data was collected.

    ______________________________________                                        TIL 714 CELLS                                                                                    Perfused                                                                      Dot-Matric                                                                            Fed-Batch                                          ______________________________________                                        Growth surface area on day 0, CM2                                                                  121        700                                           Growth surface area on day 10, CM2                                                                 121       5600                                           Total viable cell number on day 0                                                                  1.04 × 10.sup.8                                                                   8.55 × 10.sup.8                          Total viable cell number on day 10                                                                 1.12 × 10.sup.9                                                                   8.88 × 10.sup.9                          Cell viability on day 0                                                                            94%       93%                                            Cell viability on day 10                                                                           85%       86%                                            Viable cell number per CM2 on day 0                                                                8.60 × 10.sup.5                                                                   1.22 × 10.sup.6                          Viable cell number per CM2 on day 10                                                               9.26 × 10.sup.6                                                                   1.59 × 10.sup.6                          Viable cell number per ml on day 0                                                                 2.30 × 10.sup.6                                                                   1.71 × 10.sup.6                          Viable cell number per ml on day 10                                                                1.87 × 10.sup.7                                                                   2.22 × 10.sup.6                          Number of mono-cell-layers on day 0                                                                0.69      0.98                                           Number of mono-cell layers on day 10                                                               7.40      1.27                                           ______________________________________                                    

EXAMPLE 5

Cells isolated from a normal donor were cultured in a static SteriCell™bag (DuPont) for three days in serum-free Aim-V medium (Gibco)supplemented with 10 units/ml of recombinant IL-2 (rIL-2). On day 0 allcells from the bag were transferred to a 1.0μ dot-matrix vessel ofExample 1 which was then perfused with the same rIL-2 supplementedmedium for 18 days at a flow rate of 10 ml/hr. On the indicated days LAKcell activity, as measured by LU30/106 cells, was determined with a fourhour 51 Cr release assay against the human lymphoblastoid cell lineRaji. Cell counts, viability, glucose and lactic acid determination werealso performed to monitor cell survival and metabolism. The followingdata were obtained:

    __________________________________________________________________________                    Total  LU.sub.30 /                                                                        Total Lytic                                                                         Glucose                                                                            Lactate                                Device  Days                                                                             Viability                                                                          Viable Cells                                                                         10.sup.6 Cells                                                                     Units [mg/dl]                                                                            [mMolar]                               __________________________________________________________________________    Dot Matrix Bag                                                                        0  83%  9.0 × 10.sup.8                                                                 36.8 33,000                                                                              300  0                                              3  87%  3.7 × 10.sup.8                                                                 47.1 17,000                                                                              256  4.5                                            7  82%  5.3 × 10.sup.8                                                                 143.0                                                                              75,000                                                                              203  9.8                                            10 89%  5.9 × 10.sup.8                                                                 95.8 56,000                                                                              216  8.8                                            14 74%  6.1 × 10.sup.8                                                                 91.0 55,000                                                                              176  13.2                                           18 68%  7.9 × 10.sup.8                                                                 50.0 39,000                                                                               53  13.2                                   __________________________________________________________________________     Total lytic units = LU.sub.30 /10.sup.6 cells × total viable cells 

EXAMPLE 6

Cells from a normal donor were activated in Aim-V medium (Gibco) with 10units/ml rIL-2 by culture for 3 days in SteriCell™ bags (DuPont) beforethey were partitioned into two static cultures in SteriCell™ bags(DuPont) at two different concentrations and into a 0.45μ Dot Matrixvessel of Example 1, perfused at 10 ml/hr. The following data wereobtained:

    __________________________________________________________________________                    Total  LU.sub.30 /                                                                        Total Lytic                                                                         Glucose                                                                            Lactate                                Device  Days                                                                             Viability                                                                          Viable Cells                                                                         10.sup.6 Cells                                                                     Units [mg/dl]                                                                            [mMolar]                               __________________________________________________________________________    SteriCell ™ Bag                                                                    0  95%  9.0 × 10.sup.7                                                                 2.1    189 300  0                                      Low Density                                                                           3  92%  4.6 × 10.sup.7                                                                 9.5    390 284  1.8                                    Static  7  94%  5.8 × 10.sup.7                                                                 59.8 2,700 242  .6                                     Culture 10 81%  7.0 × 10.sup.7                                                                 27.2 1,300 164  13.6                                           14 40%  1.26 × 10.sup.8                                                                9.0    680  99  19.5                                   SteriCell ™ Bag                                                                    0  95%  9.0 × 10.sup.8                                                                 2.1  1,890 300  0                                      High Density                                                                          3  91%  4.56 × 10.sup.8                                                                9.1  3,700 123  17.4                                   Static  7  89%  4.32 × 10.sup.8                                                                10.0 3,400  86  18.6                                   Dot Matrix Bag                                                                        0  90%  9.0 × 10.sup.8                                                                 2.1  1,890 300  0                                              3  89%  5.5 × 10.sup.8                                                                 12.6 6,930 278  2.1                                            7  85%  7.8 × 10.sup.8                                                                 39.9 29,400                                                                              260  3.7                                            10 88%  9.7 × 10.sup.8                                                                 32.0 31,000                                                                              164  7.2                                            14 74%  1.1 × 10.sup.9                                                                 122.0                                                                              130,000                                                                              99  7.3                                    __________________________________________________________________________

EXAMPLE 7

Cells freshly collected from a normal donor were partitioned intoSteriCell™ bags (DuPont) and into a 0.45μ Dot Matrix vessel ofExample 1. The culture medium used was Aim-V (Gibco) supplemented with10 units/ml rIL-2. Flow rate in the Dot Matrix vessel was 10 ml/hr. Thefollowing data were obtained:

    __________________________________________________________________________                    Total  LU.sub.30 /                                                                        Total Lytic                                                                         Glucose                                                                            Lactate                                Device  Days                                                                             Viability                                                                          Viable Cells                                                                         10.sup.6 Cells                                                                     Units [mg/dl]                                                                            [mMolar]                               __________________________________________________________________________    SteriCells ™                                                                       0  95%  1.0 × 10.sup.8                                                                 0.0     0  300  0                                      Bag                                                                           Low Density                                                                           3  95%  7.6 × 10.sup.7                                                                 19.8 1,500 287  1.3                                    Static  6  94%  8.4 × 10.sup.7                                                                 66.1 5,500 266  3.3                                    Culture                                                                       SteriCell ™                                                                        0  95%  1.0 × 10.sup.9                                                                 0.0     0  300  0                                      Bag                                                                           High Density                                                                          3  95%  3.3 × 10.sup.8                                                                 11.4 3,700 177  11.3                                   Static  6  96%  7.7 × 10.sup.8                                                                 55.2 42,000                                                                               93  17.2                                   Culture                                                                       Dot Matrix Bag                                                                        0  95%  1.0 × 10.sup.9                                                                 0.0     0  300  0                                              3  95%  4.9 × 10.sup.8                                                                 12.2  5,900                                                                              224  6.2                                            6  95%  6.6 × 10.sup.8                                                                 66.5 43,000                                                                              212  7.6                                    __________________________________________________________________________

EXAMPLE 8

Cells from a normal donor were placed in a 1.0μ Dot Matrix vessel ofExample 1 at a high concentration and into a static SteriCell™ bag(DuPont) at a density known to yield good LAK cell activation. Cellswere cultured in Aim-V medium (Gibco). The flow rate in the Dot Matrixdevice was maintained at 10 ml/hr.

    __________________________________________________________________________                    Total  LU.sub.30 /                                                                        Total Lytic                                                                         Glucose                                                                            Lactate                                Device  Days                                                                             Viability                                                                          Viable Cells                                                                         10.sup.6 Cells                                                                     Units [mg/dl]                                                                            [mMolar]                               __________________________________________________________________________    Dot Matrix                                                                            0  100% 5.8 × 10.sup.9                                                                 0.0  0     300  0                                      At Very 3   26%   5 × 10.sup.7                                                                 0.0  0     119  19.4                                   High Density                                                                  SteriCell ™                                                                        0  100% 5.8 × 10.sup.8                                                                 0.0  0     300  0                                      Bag                                                                           Static  3   98% 3.7 × 10.sup.8                                                                 28.6 10,000                                                                              250  5                                      Culture                                                                       __________________________________________________________________________

The perfusable cell culture device of the present invention iscommercially advantageous over currently available perfused systems. Thedevice is not bulky, non-breakable and disposable. The flat surface ofthe device allows cells to spread out and stack and produces a highdensity of cells. The device also provides negligible shear to the cellsand can be operated to allow for continuous cultivation of cells. Thedevice is capable of acclimating to the temperature of the environmentfor optimal conditions and provides a sterile environment formaintenance of cells in culture. In addition, the cells can be visuallyexamined since the device is transparent and the cells can be easilyremoved from the non-adherent surface of the flow divider membrane.Having described this invention in detail, those skilled in the art willappreciate numerous modifications may be made thereof without departingfrom the spirit of the invention. Therefore, it is not intended that thescope of this invention be limited to the specific embodimentsillustrated and described. Rather, the scope is to be determined by theappended claims and their equivalents.

What is claimed is:
 1. A perfusable cell culture device comprising:(1)two liquid impermeable polymeric film sheets constructed so as todeliver oxygen and carbon dioxide at rates sufficient to maintain cellsin culture joined together at their peripheral edges in a sealingmanner, one of the sheets defined as an upper polymeric layer and theother of the sheets defined as a lower polymeric layer; (2) a gas andliquid permeable flow divider membrane affixed between said upperpolymeric layer and said lower polymeric layer and affixed to said lowerpolymeric layer in such a way so as to provide channels for fluiddistribution with the pressure drop across the flow divider membranebeing significantly greater than pressure drop through the channelsthereby providing substantially even pressure across the flow dividerarea and allowing the culture medium to flow uniformly over a largesurface area; (3) an inlet port in said lower polymeric layer throughwhich culture medium can be introduced between said lower polymericlayer and said flow divider membrane; (4) said upper polymeric layer andsaid flow divider membrane being arranged with respect to each other soas to form a chamber between said upper polymeric layer and said flowdivider membrane through which growth medium can be perfused at acontrolled rate uniformly across the surface of the flow dividermembrane and in which cells are cultured; and (5) a port in said upperpolymeric layer through which cells may be placed into the cell culturedevice, cultured cells may be removed from said cell culture device, andperfused nutrients may be collected from said cell culture device.
 2. Aperfusable cell culture device according to claim 1 wherein the materialfor the upper and lower polymeric layers is selected from the groupconsisting of a copolymer of ethylene and an alpha olefin,polytetrafluoroethylene, and fluorinated ethylenepropylene copolymer,said materials being permeable to oxygen and carbon dioxide at ratessufficient to maintain cells in culture.
 3. A perfusable cell culturedevice according to claim 1 wherein the flow divider membrane isthermally sealed to the lower polymeric layer with an equally spacedpattern forming the channel for fluid distribution.
 4. A perfusable cellculture device according to claim 3 wherein the equally spaced patternis a dot matrix pattern.
 5. A perfusable cell culture device accordingto claim 1 wherein the flow divider membrane is joined to the lowerpolymeric layer with an equally spaced pattern forming the channels forfluid distribution.
 6. A perfusable cell culture device according toclaim 5 wherein the equally spaced pattern is a dot matrix pattern.
 7. Aperfusable cell culture device according to claim 5 wherein the meanpore size within the flow divider membrane is between 0.45 um and 1.0 umin diameter.
 8. A perfusable cell culture device according to claim 1wherein the gas and liquid permeable flow divider membrane is expandedpolytetrafluoroethylene (PTFE).
 9. A perfusable cell culture deviceaccording to claim 8 wherein the expanded polytetrafluoroethylene (PTFE)material is coated with a water soluble polymer whereby the expandedpolytetrafluoroethylene (PTFE) material is rendered more hydrophilicwith reduced contact angle.
 10. A perfusable cell culture deviceaccording to claim 10, wherein the water soluble polymer ispolyvinylpyrroidone.